(1) Field of the Invention
This invention relates to a process for the production of dried earthworm powder, as well as antihyperlipemic, antidiabetic and blood pressure regulator (antihypertensive and/or antihypotensive) preparations containing the dried earthworm powder as the active ingredient. More particularly, it relates to a novel and improved process for the production of pharmaceutically acceptable dried earthworm powder which has not only excellent antihyperlipemic, antidiabetic (or hypoglycemic), antihypertensive and/or antihypotensive effects but also a high degree of safety, as well as antihyperlipemic, antidiabetic and blood pressure regulator (antihypertensive and/or antihypotensive) preparations containing the dried earthworm powder as the active ingredient. Thus, the dried earthworm powder produced by the process of the present invention may be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions which are useful for the treatment or prevention of hyperlipemia, diabetes, hypertension and hypotension in mammals and, in particular, human beings.
(2) Description of the Prior Art
It is recognized that hyperlipemia, along with hypertension, diabetes and smoking, is an important causative factor in arteriosclerosis, and a variety of antihyperlipemic drugs comprising synthetic organic compounds have been developed and used for the treatment and prevention of hyperlipemia. Typical examples thereof are clofibrate and nicomol. However, it is known that clofibrate and its derivatives may frequently cause muscle pain, hepatic functional disorders and gallstones formation. It is also known that nicomol has side effects such as facial suffusion and gastrointestinal disorders. Moreover, it has been reported that clofibrate may produce hepatoma in animals [D. J. Svoboda et al., Cancer Res., Vol. 39, p. 3419 (1979)].
In addition to the above-described problem of safety, special attention is also paid to the effect and pharmacology of antihyperlipemic drugs. Specifically, with the recent progress of research on the metabolism of lipids and, in particular, the functions of serum lipoproteins as serum lipid carriers, much importance has come to be attached not only to the ability of a drug to reduce the concentration of lipids in the serum, but also to its action on lipoproteins.
Serum cholesterol, together with triglycerides (hereinafter referred to as TG), phospholipids (hereinafter referred to as PL) and apoproteins, forms lipoproteins. According to density, these lipoproteins are divided into very low density lipoprotein (hereinafter referred to as VLDL), low density lipoprotein (hereinafter referred to as LDL) and high density lipoprotein (hereinafter referred to as HDL). Among these lipoproteins, VLDL and LDL are thought to induce arteriosclerosis. In contrast, HDL functions to participate in the transport of cholesterol from peripheral blood vessels to the liver, the formation of cholesterol ester and the catabolism of TG, and is believed to have the effect of preventing and retracting arteriosclerosis.
Accordingly, in the future development of an antihyperlipemic drug, importance should be attached not only to its effect of reducing the level of total cholesterol (hereinafter referred to as TC) in the serum, but also to the type of lipoprotein whose cholesterol it can act. In particular, it would be desirable to develop a drug not only having the effect of reducing the level of cholesterol in LDL (hereinafter referred to as LDL-C) and elevating the level of cholesterol in HDL (hereinafter referred to as HDL-C), but also having the effect of lowering the arteriosclerosis index (hereinafter referred to as AI) which is calculated from the formula: (TC - HDL-C)/HDL-C.
Conventionally, organic synthetic compounds such as sulfonylurea compounds and biguanide compounds have been widely used as oral antidiabetic drugs. Generally, antidiabetic drugs are medicines used to normalize the metabolism in the morbid state, and not medicines capable of curing diabetes itself. In the case of diabetic coma or juvenile diabetes, oral antidaibetic drugs are ineffective and insulin alone is effective. In several forms of diabetes in which insulin is mandatory (for example, diabetes associated with ketoacidosis or serious infection), oral antidiabetic drugs are ineffective. The chief indication for the use of oral antidiabetic drugs is maturity-onset diabetes which cannot be fully controlled by alimentotherapy. Also in this case, such oral antidiabetic drugs tend to produce side effects such as hypoglycemia, hepatic functional disorders and anorexia, and must be used under the rigid control of a physician. Thus, there is an eager demand for an antidiabetic drug having no side effects.
In recent years, there are a large numbers of patients with hypertension or hypotension, irrespective of age. In order to treat such patients, hypotensors are often administered to hypertensive patients and, though not mandatory, hypertensors (or pressors) are often administered to hypotensive patients.
Especially with the recent increase in the number of hypertensive patients, a wide variety of remedies for hypertension (or hypotensors) have been being developed and used. Once hypertensive patients begin to take a hypotensor, discontinuance of its use may cause the symptoms to become worse than before. Accordingly, it is often necessary to take the drug continuously for a long period of time. Also in the case of hypotensive patients to whom a drug is administered, its administration is often continued for a long period of time. Thus, there is an eager demand for a drug having no side effects.
For example, hydralazine known to be a hypotensor has the effect of dilating peripheral blood vessels and exhibits excellent hypotensive activity. However, it may cause tachycardia as a side effect, so that it is used in combination with a .beta.-blocker. Generally, hypotensors and hypertensors are used for a long of time, so that it would be highly desirable to provide such drugs having no side effects.
Especially in the Oriental countries, earthworms (also called "dilong") have been used as a drug from remote antiquity. The following pharmacological effects of easthworms have been reported in the literture.
(1) Shinryu Ofuchi ["Mimizu-to-Jinsei (Earthworms and Human Life)", Maki Shobo, Oct. 30, 1947, pp. 223-226] and Nikiji Hatai ["Mimizu (Earthworms), Reprinted Edition", Scientist Co., Apr. 30, 1980, pp. 160-163] have reported that earthworms have a variety of pharmacological activities, i.e., they are effective in reducing the size of vesical calculi and eliminating them from the body, in the treatment of jaundice, and as a parturifacient, restrative, hair grower, tonic and an antipyretic. On the other hand, they have also reported some toxic actions of earthworms. That is, earthworm poison injures the nervous systems and causes hemolysis (or the destruction of red blood cells).
(2) The following description is found in "Pharmaceutical Dictionary of the People's Republic of China" edited by the Pharmaceutical Dictionary Editing Committee, the Department of Hygienics, the People's Republic of China (1977 Edition, Part I, pp. 197-198).
Conventionally, there are two types of dilong. One of them is wide dilong (Lumbricus kwangtungesis) that is produced by cutting the body of each earthworm open, washing off the guts and soil, and drying it in the sun, in the shade or at low temperature. The other is soil dilong (Lumbricus nativus) that is produced by by killing earthworms in ash from plants, freeing them of ash, and drying them in the sun, in the shade or at low temperature. The dried earthworm so produced have soil within the body thereof. It is reported therein that these two types of dilong are used in a daily dose of 4.5 to 9 g as an antipyretic, anticonvulsant, circulation promoter, remedy for hemiplegia, articular analgesic, diuretic, antiasthmatic and antihypertensive.
(3) It is described in "Our Chinese Medicine Series 3: Dilong and Cuttlebone--Scientific Research in China" (Matsuura Yakugyo K. K., p. 7) that dilong tincture (i.e., an ethyl alcohol extract of dilong) has an hypotensive effect.
(4) It has been reported by Takuo Okuda ("Encyclopedia of Natural Medicines", Hirokawa Shoten, Apr. 15, 1986, p. 215) that dilong is being used as an antipyretic, analgesic, diuretic and antidote.
(5) Mamotu Tanaka [Hokkaido Medical Journal, Vol. 24, pp. 18-24 (1949)] has reported the results of an experiment with earthworms. Specifically, small pieces of dried earthworms were freed of soil and then extracted with boiling water. After the resulting extract was concentrated, ethyl alcohol was added thereto and the precipitated material (lumbrofebrin) was dissolved in Ringer's solution. When this solution was intravenously injected into an anesthetized cat, a sudden fall in blood pressure was caused. In addition, acceleration of blood coagulation was observed in proportion to the shock.
(6) Kenjiro Ikawa [Yamaguchi Igaku, Vol. 9, pp. 571-576 (1960)] has reported that test solutions were prepared by extracting dilong with physiological saline, or by extracting dilong with ethyl alcohol or acetone, evaporating the extract to dryness and dissolving the residue in physiological saline. When each of these test solutions was intravenously injected into a mature rabbit, a fall in blood pressure was observed.
(7) It is reported in "Enzyclopedia of Chinese Medicines, Volume Two" edited by the Koso New Medical Institute (Shanghai Scientific and Technical Publishing Company, 1980, p. 2112) that, when wide dilong tincture, a suspension of dried earthworm powder, a hot water infusion of earthworms, or a decoction of earthworms was administered to anesthetized dogs, big rats, cats, or mice with chronic renal hypertension, a slow and long-lasting hypetnesive effect was noted. When dilong extract was intravenously injected into anethetized dogs or cats, a rapid hypotensive effect appeared. However, it is also reported that they were ineffective when administered orally and when used in clinical trials. Moreover, it is reported in the same book (p. 2114) that essential hypertension could be effectively controlled by oral administration of dilong. To this end, 10 ml of 40% dilong tincture (prepared by steeping 40 g of dilong in 100 ml of 60% ethyl alcohol) was given three times a day [i.e., in a daily dose of 12 g of dilong (as calculated by the present inventors)] and this treatment was continued for 30 to 60 days. For those who could not drink the tincture, pills were prepared from pure dilong powder mixed with water (and a small amount of filler, and 3 to 4 g of the pills were given three times a day [in a daily dose of 9 to 12 g of dilong (as calculated by the present inventors)]. Furthermore, it is reported that essential hypertension could also be effectively controlled by administering 2 ml of dilong B.sub.1 liquid (prepared by removing hypoxanthine from dilong with the aid of HgCl.sub.2 and then isolating hypotensive components therefrom by means of an ion-exchange resin) three times a day [in a daily dose of 24 g of dilong (as calculated by the present inventors)].
Conventional methods for producing the dried products of dried powder of earthworms are roughly divided into the following three types.
(i) The method in which the body of each earthworm is cut open, freed of its contents (i.e., the guts and soil), and then dried in the sun, in the shade or at low temperature (usually 50.degree. C. or below).
(ii) The method in which earthworms are killed by placing them in ash from plants or charcoal, freed of any soil, and then dried in the sun, in the shade or at low temperature (usually 50.degree. C. or below) to obtain dried earthworms having soil therein.
(iii) The method in which earthworms are freed of the soil present in their body and then dried by placing them in ash from plants or charcoal.
In case of need, the dried earthworms thus obtained are reduced to powder and used. The above-described methods are simple and economical ones and has the advatage that they can easily be carried out at home. However, if the dried earthworms (or dried earthworm powder) produced by these methods are preserved in an open state either in a referigerator at 0.degree. to 5.degree. C. or in a room at 5.degree. to 45.degree. C., they will become moldy within about 6 months and cannot be used any longer. Even if they are preserved in a well-closed state, they will become moldy within a year.
Where dried earthworms having soil within the body thereof, as produced by the aforesaid method (ii), or earthworms dried in ash from plants or charcoal, as produced by the aforesaid method (iii), are used as a medicine, it is common practice to extract them with hot water or decoct them in boiling water, filter the resulting extract or decoction, and take the filtrate. In particular, it is seldom that the dried earthworms produced by method (ii) is used in the form of dilong tincture, powder or pills.
The dried earthworms produced by method (i) are frequently used as a hot water infusion or decoction, or in the form of dilong tincture, dilong powder or pills (prepared from dilong powder mixed with a small amount of water or a small amount of filler). In the case of methods (ii) and (iii), the yield of dried earthworms having a moisture content of 10 to 16% is 5 to 19% based on the living earthworms used as the raw material.
Recently, Yoichi Ishii who is one of the present inventors has proposed a health food comprising, as principal components, the proteins and lipids derived from earthworms, and a process for producing the same [Japanese Patent Laid-Open No. 216572/'84 (date of laying open: Dec. 6, 1984)]. This process is an excellent one for the purpose of producing dried earthworm powder as a health food. However, as a method for producing dried earthworm powder for use as antihyperlipemic, abtidiabetic and blood pressure regulating drugs, the aforesaid process is not satisfactory from the viewpoint of efficacy. Specifically, if an external action is exerted on living earthworms to eliminate the soil present in the body thereof, it is impossible to remove the soil selectively. It has been found that, even if the utmost care is taken, the internal organs and body fluids containing the major portion of components highly important from the viewpoint of medicinal effects is at least partially removed together with the soil, resulting in insufficient efficacy. Moreover, the yield is as low as 10 to 19% based on the living earthworms used as the raw material. Furthermore, it is a great disadvantage that, since the final stage of vacuum drying is carried out at 80.degree. C. under a vacuum of 0.3 torr for a long period of 20 hours or more, the enzymes present in the dried earthworm powder and playing an important role in the manifestation of medicinal effects are at least partially destroyed or inactivated. Accordingly, the antihypertensive effect of the dried earthworm powder produced by the process of Japanese Patent Laid-Open No. 216572/'84 has been found to be about 50% of that of the dried earthworm powder produced by the process of the present invention.
Recently, Hisahi Mihara, who is one of the present inventors, and his collaborators have demonstrated that the fibrinolytic substance derived from earthworms is an enzyme protein which has an optimum pH of 8 to 10; is stable in the pH range of 5 to 10; is inhibited by Trazirol (trade name), Transamine (trade name), soybean trypsin inhibitor and serum; has plasminogen-activating and fibrynolytic effects; and has no fibrinogenolytic effect.
A crude enzyme protein fraction obtained by extraction of an earthworm with an aqueous medium, and a process for the preparation of a fibrinolytic substance by purifying the crude enzyme enzyme protein fraction are disclosed in Japanese Patent Laid-Open No. 148824/'83 (filed Feb. 27, 1982) and its corresponding foreign patent applications including U.S. patent application Ser. No. 470,394 (filed Feb. 28, 1983), U.K. Pat. Appln. No. 8305359 (filed Feb. 25, 1983), Italian pat. appln. No. 477954A (filed Feb. 25, 1983), French Pat. Appln. No. 03165 (filed Feb. 25, 1983), German Pat. Appln. No. P3306944.1 (filed Feb. 28, 1983) and Canadian Pat. Appln. No. 422034 (filed Feb. 21, 1983).
Furthermore, Hisashi Mihara and his collaborators have derived six novel proteases from earthworms, as disclosed in Japanese Patent Laid-Open No. 63184/'84 (filed Oct. 2, 1982), and have developed a thrombolytic preparation comprising these proteases as the active ingredients, as disclosed in Japanese Patent Laid-Open No. 184131/'84 (filed Mar. 31, 1983). These inventions are also disclosed in their consolidated forein patent applications including Korean Pat. Appln. No. 2990 (filed Jul. 30, 1983), Au. Pat. Appln. No. 16293 (filed Jun. 27, 1983), Ca. Pat. Appln. No. 431387 (filed Jun. 28, 1983), Dk. Pat. Appln. No. 3008 (filed Jun. 29, 1983), Ep. Pat. Appln. No. 83106288.0 (filed Jun. 28, 1983), Es. Pat. Appln. No. 523754 (filed Jun. 30, 1983), Fi. Pat. Appln. No. 832383 (filed Jun. 29, 1983), No. Pat. Appln. No. 2399 (filed Jun. 30, 1983), Ph. Pat. Appln. No. 29151 (filed Jun. 30, 1983), Tw. Pat. Appln. No. 7211983 (filed Jun. 18, 1983) and U.S. patent application Ser. No. 508,163 (filed Jun. 27, 1983).
The present inventors made an investigation of the literature and obtained the following results:
(1) In the literature, no mention can be found of the antihyperlipemic effect of pharmaceutical preparations containing dried earthworm powder or dilong as the active ingredient. Still less, it is not reported therein that dried earthworm powder has not only the effect of reducing the serum TC level, but also the effect of reducing the serum LDL-C level and elevating the serum HDL-C level to cause a lowering of AI, and that dried earthworm powder is a highly safe drug which does not producing side effects such as hepatic hypertrophy and hepatic functional disorders.
(2) In the literature, no mention can be found of the antidiabetic or hypoglycemic effect of pharmaceutical preparations containing dried earthworm powder as the active ingredient.
That is, it is not reported in the literature that, in rats with diabetes experimentally induced by alloxan, the blood sugar level can be significantly reduced by administering dried earthworm powder thereto. As will be described later, capsules containing dried earthworm powder, in combination with alimentotherapy, were administered to diabetic patients for 4 to 9 months. Thus, in the case of patients with a mild or moderate degree of diabetes, the blood sugar level began to lower after 2 or 3 months of treatment, and returned to the normal value of healthy persons after 4 months of treatment. Such as excellent effect cannot be found in the literature.
(3) In the literature, no mention can be found of the antihypotensive effect of pharmaceutical preparations containing dried earthworm powder as the active ingredient. Moreover, it is not reported therein that dried earthworm powder has a blood pressure regulating effect, i.e., the administration of dried earthworm powder to patients with hypertension and/or hypotension restores their maximum and minimum blood pressures to normal.